Passive epidemiological surveillance in wildlife in Costa Rica identifies pathogens of zoonotic and conservation importance

Epidemiological surveillance systems for pathogens in wild species have been proposed as a preventive measure for epidemic events. These systems can minimize the detrimental effects of an outbreak, but most importantly, passive surveillance systems are the best adapted to countries with limited resources. Therefore, this research aimed to evaluate the technical and infrastructural feasibility of establishing this type of scheme in Costa Rica by implementing a pilot program targeting the detection of pathogens of zoonotic and conservation importance in wildlife. Between 2018 and 2020, 85 carcasses of free-ranging vertebrates were admitted for post-mortem and microbiology analysis. However, we encountered obstacles mainly related to the initial identification of cases and limited local logistics capacity. Nevertheless, this epidemiological surveillance scheme allowed us to estimate the general state of health of the country’s wildlife by establishing the causes of death according to pathological findings. For instance, 60% (51/85) of the deaths were not directly associated with an infectious agent. Though in 37.6% (32/85) of these cases an infectious agent associated or not with disease was detected. In 27.1% (23/85) of the cases, death was directly related to infectious agents. Furthermore, 12.9% (11/85), the cause of death was not determined. Likewise, this wildlife health monitoring program allowed the detection of relevant pathogens such as Canine Distemper Virus, Klebsiella pneumoniae, Angiostrongylus spp., Baylisascaris spp., among others. Our research demonstrated that this passive surveillance scheme is cost-effective and feasible in countries with limited resources. This passive surveillance can be adapted to the infrastructure dedicated to monitoring diseases in productive animals according to the scope and objectives of monitoring wildlife specific to each region. The information generated from the experience of the initial establishment of a WHMP is critical to meeting the challenges involved in developing this type of scheme in regions with limited resources and established as hotspots for emerging infectious diseases.

I am attaching the revised version of our manuscript: "Passive epidemiological surveillance in wildlife in Costa Rica identifies pathogens of zoonotic and conservation importance" by Fernando Aguilar-Vargas and Tamara Solorzano-Scott1 et al., which we would like to resubmit for publication as a Research Article to PLoS ONE.
We want to acknowledge all the reviewers' comments since they have helped us shape our work and improve the manuscript. We have paid attention to all the concerns raised by both reviewers, made the changes when required, and highlighted all changes in yellow color in the text.
We appreciate the editor and referees' time and efforts in reviewing this manuscript and believe that the revised version will meet the journal publication requirements.
Sincerely yours, Dr. Alejandro Alfaro-Alarcón PhD. Department of Anatomic Pathology National University of Costa Rica Head of Department

Reviewer 1
In their first submission, the authors only presented their results without proposing a feasible and standardized method to implement this tool. In this version, they provide more detail (exemplified in figure 1), but I am afraid that their suggestions are very country specific. If this manuscript is accepted, I suggest that it would be under the study type "Methods, software, databases, and tools", which should meet the criteria of utility, validation and availability (see https://journals.plos.org/plosone/s/submission-guidelines#loc-methodssoftware-databases-and-tools), and should be developed more broadly to propose a scheme for other countries with similar challenges. Although, their epidemiological findings are interesting by themself, they are not the real focus of the paper, but rather are the evidence to stress the need to implement their proposed passive surveillance program in a sustainable manner with the tools that are already available. However, it seems like the authors shift the focus of their paper in presenting their findings. As an example, tables 3, 4 and 5 could be supplemental materials.
R/ We thank reviewer #1 for the constructive and helpful comments. The reviewers' suggestion to standardize a general passive wildlife surveillance scheme for countries with limited resources is an exciting objective. However, we consider that that idea exceeds the scope and intentions of this research. This manuscript aims to communicate our experience of what could be the establishment of a passive epidemiological surveillance system in the wildlife of Costa Rica as a model. This study is a pilot effort with the intention that the scientific community of other countries can visualize what aspects should be considered when aspiring for a surveillance system for wildlife animals. We believe that the current scope of this study can be helpful to countries pursuing this goal.
We also want the manuscript to illustrate aspects such as the type of agents detected and how to approach their diagnosis based on the available diagnostic capacities. This is essential in countries with low income dedicated to epidemiological surveillance of wildlife. In addition, we want to highlight the importance of linkage among human, animal, and wildlife health authorities and higher education institutions under the one health approach. This is based on the urgent need to raise awareness of the benefits of this type of disease monitoring for the countries and its regional and global impact.
Based on this study's limitations, we have recognized the weaknesses in lines: #357-365. However, the proposed program is limited to Costa Rica's resources and infrastructure and does not apply to every country. Therefore, we emphasize that it is important to start evaluating the implementation of these programs in regions where disease surveillance in wildlife is minimal based on experiences like ours. Furthermore, although it is necessary to standardize methods and techniques for monitoring pathogens in wildlife, the development of pilot schemes allows sharing experiences with other programs already installed.
In tune with the European Wildlife Disease Association and OIE, although efforts have been made to expand and unify surveillance programs dedicated to wildlife, significant variations persist. There are still variations in the scope, scale, and capacity for establishing surveillance schemes in many regions. These limitations are mentioned in lines: #372-375.
We are confident that in future studies, we will be able to compare our scheme with other WHMPs in the region to contribute to building these systems in other regions. In addition, other upcoming experiences would help standardize and optimize monitoring systems in the scientific community. Based on the reasons mentioned above and by recognizing the limitations of our study in the manuscript, we believe that the study should maintain the initially conceived structure and leave for future studies the pertinent approach proposed by reviewer 1.
Finally, I found the manuscript difficult to follow. Occasionally, the sentences read awkward (e.g., Lines 50-53), had typos (e.g., Line 55) or wrong terms (e.g., "academy" in Lines 312-314), were grammatically incorrect (e.g., Line 80-82 and Line 95-97), or lacked a clear structure (e.g., Lines 108-112). The authors should do some intense editing to their manuscript, with a more concise language, and, if available, consider asking for Englishproof reading services. R/We apologize for these mistakes. The English language and grammar were revised throughout the text, and the manuscript was improved to make it more concise and fluent.

Reviewer #2:
We would like to thank reviewer #2 for the positive comments I recommend the use the following categories: metazoan parasites, protozoan parasites. This was modified as requested Please, double check the grammar of the text, mainly in the introduction, in general, the English could be a little more fluent. However, I emphasize that this does not affect at all the understanding of the ideas they expose. The English language was revised throughout the text.
Minor comments: Line 5: delete of (repeated) This was corrected Line 197: change pulmonary parasitic worms to lungworm. This was corrected